改良分子信标-实时PCR快速检测产单核李斯特菌

Rapid simultaneous detection of Listeria monocytogenes using modified molecular beacons and real-time PCR

目的:建立改良分子信标-实时PCR检测产单核李斯特菌(LMO)的快速方法,应用于食品中LMO的污染状况调查及食物中毒快速诊断。方法:根据GenBank公布的LMO hlyA基因的保守序列,设计引物和改良分子信标探针,建立改良分子信标-实时PCR检测体系,应用于食品中LMO检测。结果:改良分子信标-实时PCR反应体系DNA灵敏度为110 fg,菌液灵敏度为99 cfu/m l或4 cfu/PCR反应体系,无交叉反应。以此反应体系检测28株LMO,均出现特异的荧光信号。上述方法可将检测时间由原来的至少4 d缩短至1 d。对228份食品进行LMO检测,8份增菌液LMO实时荧光PCR阳性,其中6份LMO细菌培养阳性。结论:改良分子信标-实时PCR反应体系快速、灵敏度高,特异性强,能提高LMO的检出率和准确性,可应用于LMO食品污染状况调查及食物中毒的快速诊断。

Objective ： Rapid detection of Listeria monocytogenes （LMO） using modified molecular beacons and real - time PCR was developed. The established method was applied to an investigation of the infectious status of Listeria monocytogenes in food and LMO food poisoning. Methods：One set of primers was designed based on the core sequence of hlyA gene published on GenBank to detect LMO. The corresponding modified molecular beacons labeled with FAM were designed. The modified molecular beacons and real - time PCR assay were applied to detect the LMO in food. Results： For the modified molecular beacons - based real - time PCR assay, the sensitivity achieved was 110 fg or 4 cfu/PCR reaction. There was no cross -reaction with other bacteria as control. The real - time PCR assay was used to detect 28 LMO strains, no false signals were observed. The detection time was reduced from at least five days to only one day. A total of 228 food poisoning samples were tested, 8 were found LMO positive by real - time PCR. Among the tested positive samples, 6 were LMO positive by modified traditional method. The detection rate of LMO in food in Shenzhen was 2. 63%. Conclusion：The modified molecular beacons - based real - time PCR assay is rapid, sensitive and specific. It could be applied to the rapid diagnosis of LMO food poisoning and investigation of the infectious status of it in food.